LOCUS       sequence-14176-e        5979 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5979)
  AUTHORS   Waldner C, Rempel O, Schutte F, Yanik M, Solomentsew N, Ryffel GU
  TITLE     Double conditional human embryonic kidney cell line based on FLP and
            PhiC31 mediated transgene integration.
  JOURNAL   BMC Res Notes. 2011 Oct 18;4:420.
  PUBMED    22008483
REFERENCE   2  (bases 1 to 5979)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "BMC Res 
            Notes."; date: "2011-10-18"; volume: "4"; pages: "420"
COMMENT     Sequence Label: pINT-PuroDDEYFP
FEATURES             Location/Qualifiers
     source          1..5979
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      6..15
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(12..14,15..335)
                     /codon_start=1
                     /product="destabilization domain that can be stabilized by 
                     Shield1 in the ProteoTuner(TM) system"
                     /label=DD
                     /note="L106P mutant of FKBP12"
                     /translation="MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNK
                     PFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVEL
                     LKPE"
     CDS             join(342..344,345..1058)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(384..405)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(645..664)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     992..1013
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1088..1222
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1335..1355)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1335..1353)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1374..1390)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1374..1390)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1387..1409)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1398..1414
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(1422..1428,1429..1446,1447..1452))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1467..1488
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1605..1622)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1776..2364)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1856..1875)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2535..3326,3327..3395))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     3158..3177
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3396..3500)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3526..3981)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3663..3684)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3875..3894
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    4058..4091
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     CDS             4092..4688
                     /codon_start=1
                     /gene="pac from Streptomyces"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="TEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERV
                     TELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAA
                     QQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSA
                     PRNLPFYERLGFTVTADVECPKDRATWCMTRKPGA"
     primer_bind     4585..4605
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    4712..4846
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        4916..5900
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
     promoter        5935..5953
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     5935..5952
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gatccgccac catgggagtg caggtggaaa ccatctcccc aggagacggg cgcaccttcc
       61 ccaagcgcgg ccagacctgt gtggtgcact acaccgggat gcttgaagat ggaaagaaag
      121 tcgattcctc ccgggacaga aacaagccct ttaagtttat gctaggcaag caggaggtga
      181 tccgaggctg ggaagaaggg gttgcccaga tgagtgtggg tcagagagcc aaactgacta
      241 tatctccaga ttatgcctat ggtgccactg ggcacccagg catcatccca ccacatgcca
      301 ctctcgtctt cgatgtggag cttctaaaac cggaagaatt cgtgagcaag ggcgaggagc
      361 tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt
      421 tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca
      481 tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ttcggctacg
      541 gcctgcagtg cttcgcccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg
      601 ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca
      661 agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg
      721 gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca
      781 gccacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggtg aacttcaaga
      841 tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc
      901 ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagctac cagtccgccc
      961 tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg
     1021 ccgggatcac tctcggcatg gacgagctgt acaagtaatc tagaactata gtgagtcgta
     1081 ttacgtagat ccagacatga taagatacat tgatgagttt ggacaaacca caactagaat
     1141 gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct attgctttat ttgtaaccat
     1201 tataagctgc aataaacaag ttaacaacaa caattgcatt cattttatgt ttcaggttca
     1261 gggggaggtg tgggaggttt tttaattcgc ggccgcggcg ccaatgcatt gggcccggta
     1321 cccagctttt gttcccttta gtgagggtta attgcgcgct tggcgtaatc atggtcatag
     1381 ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc
     1441 ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc
     1501 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     1561 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg
     1621 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     1681 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     1741 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     1801 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     1861 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     1921 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     1981 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     2041 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     2101 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     2161 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca
     2221 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     2281 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     2341 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     2401 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     2461 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     2521 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     2581 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     2641 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     2701 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     2761 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     2821 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     2881 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     2941 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     3001 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     3061 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     3121 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     3181 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     3241 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     3301 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     3361 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     3421 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     3481 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctaaatt gtaagcgtta
     3541 atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg
     3601 ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg
     3661 ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa
     3721 aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg
     3781 ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt
     3841 gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg
     3901 ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta
     3961 atgcgccgct acagggcgcg tcccattcgc cattcaggct gcgcaactgt tgggaagggc
     4021 gatcggtgcg ggcctcttcg ctattacgcc agtcgaggtg ccagggcgtg cccttgggct
     4081 ccccgggcgc gaccgagtac aagcccacgg tgcgcctcgc cacccgcgac gacgtcccca
     4141 gggccgtacg caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg
     4201 atccggaccg ccacatcgag cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg
     4261 ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca
     4321 cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc atggccgagt
     4381 tgagcggttc ccggctggcc gcgcagcaac agatggaagg cctcctggcg ccgcaccggc
     4441 ccaaggagcc cgcgtggttc ctggccaccg tcggcgtctc gcccgaccac cagggcaagg
     4501 gtctgggcag cgccgtcgtg ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg
     4561 ccttcctgga gacctccgcg ccccgcaacc tccccttcta cgagcggctc ggcttcaccg
     4621 tcaccgccga cgtcgagtgc ccgaaggacc gcgcgacctg gtgcatgacc cgcaagcccg
     4681 gtgcctgaga attcgtgagt cgtattacgt agatccagac atgataagat acattgatga
     4741 gtttggacaa accacaacta gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga
     4801 tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttaaca acaacaattg
     4861 cattcatttt atgtttcagg ttcaggggga ggtgtgggag gttttttaat tcgtcgacca
     4921 tagccaattc aatatggcgt atatggactc atgccaattc aatatggtgg atctggacct
     4981 gtgccaattc aatatggcgt atatggactc gtgccaattc aatatggtgg atctggaccc
     5041 cagccaattc aatatggcgg acttggcacc atgccaattc aatatggcgg acttggcact
     5101 gtgccaactg gggaggggtc tacttggcac ggtgccaagt ttgaggaggg gtcttggccc
     5161 tgtgccaagt ccgccatatt gaattggcat ggtgccaata atggcggcca tattggctat
     5221 atgccaggat caatatatag gcaatatcca atatggccct atgccaatat ggctattggc
     5281 caggttcaat actatgtatt ggccctatgc catatagtat tccatatatg ggttttccta
     5341 ttgacgtaga tagcccctcc caatgggcgg tcccatatac catatatggg gcttcctaat
     5401 accgcccata gccactcccc cattgacgtc aatggtctct atatatggtc tttcctattg
     5461 acgtcatatg ggcggtccta ttgacgtata tggcgcctcc cccattgacg tcaattacgg
     5521 taaatggccc gcctggctca atgcccattg acgtcaatag gaccacccac cattgacgtc
     5581 aatgggatgg ctcattgccc attcatatcc gttctcacgc cccctattga cgtcaatgac
     5641 ggtaaatggc ccacttggca gtacatcaat atctattaat agtaacttgg caagtacatt
     5701 actattggaa gtacgccagg gtacattggc agtactccca ttgacgtcaa tggcggtaaa
     5761 tggcccgcga tggctgccaa gtacatcccc attgacgtca atggggaggg gcaatgacgc
     5821 aaatgggcgt tccattgacg taaatgggcg gtaggcgtgc ctaatgggag gtctatataa
     5881 gcaatgctcg tttagggaac cgccattctg cctggggacg tcggagcaag cttgatttag
     5941 gtgacactat agaatacaag ctacttgttc tttttgcag
//
